Purpose: The aim of this study was to validate the sterility testing of tissue samples according to the “Method suitability test” defined by the European Pharmacopoeia (EP, chapter 2.6.1), using the MEB buffer (AL.CHI.MI.A. S.r.l.) for extraction of microorganisms from tissue samples and RESEP (AL.CHI.MI.A. S.r.l.) for elimination of antimicrobials before direct inoculation of growth media.

Materials and methods: Samples consisting of one gram of sterile porcine aortic valve were immersed in BASE.128 (AL.CHI.MI.A. S.r.l.) at 4°C for 24 h to simulate the tissue decontamination process with an antibiotic cocktail. The samples were then contaminated with 10-100 cfu of EP reference strains (Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Bacillus subtilis, Aspergillus niger, Clostridium sporogenes), and introduced in a vial containing MEB extraction buffer and stirred at room temperature for 20 min in order to extract microorganisms from the tissues. The buffer was then treated with RESEP syringe for removal of antimicrobial residues and inoculated in Tryptic Soy Broth (TSB) or Thioglycolate (TG). The turbidity of the growth media was determined visually after 5 days of incubation at 22°C (TSB) or 33°C (TG).

Results: MEB buffer extracted the whole 10-100 cfu of all tested microorganisms from aortic valve samples. All microorganisms showed growth in TG or TSB media after RESEP treatment indicating vitality and absence of BASE.128 antimicrobial residues. Turbidity of growth media was detected within 5 days after inoculation in all tested conditions.

Conclusions: The sterility test of the tissue samples, including the extraction of microbial contaminants from tissues using MEB buffer and removal of antimicrobials using RESEP, before direct inoculation, was successfully validated according to the “Method Suitability Test” of the European Pharmacopoeia (chapter 2.6.1.).

Background: The aim of the study was to investigate the decontamination of human donor larynx (tissue-engineered for laryngeal replacements) with medical device BASE.128 (ALCHIMIA S.r.l) in comparison with standard decontamination procedure.

Methods: Laryngeal tissues from two donors were divided in halves and decontaminated with BASE.128 at 22°C and BASE.128 at 37°C overnight. The control halves of both tissues were disinfected with standard procedure using 20% chlorhexidine for 5 minutes at room temperature. Tissues were tested microbiologically, and any microorganism still present at the end of the process was isolated and further investigated in BASE.128 time-kill studies at 37°C.

Results: Standard chlorhexidine disinfection procedure and decontamination with BASE.128 at 22°C overnight resulted inefficient in elimination of C. albicans and S. oralis from the first donor larynx.
However, the time-kill studies showed 5-6 log elimination of both isolated strains, after treatment with BASE.128 at 37°C for 6h.
The second tissue, initially contaminated by Citrobacter koseri, was completely decontaminated by BASE.128 at 37°C overnight, while it still resulted contaminated after standard disinfection with 20% chlorhexidine.

Conclusion: An overnight decontamination with BASE.128 at 37°C was effective in decontaminating laryngeal tissue. More tissues will be tested in order to confirm the results and validate the procedure.

Background: Decontamination of umbilical cord is a critical phase before cryopreservation. We investigated the decontamination efficacy of the BASE.128 against the most frequent umbilical cord contaminants by an in vitro time-kill study.

Methods: F. magna (ATCC 29328), P. melaninogenica (ATCC 25845), and umbilical tissue clinical isolates E.coli, E. faecalis, and B. fragilis were cultivated under optimal growth conditions. Resistance to the antibiotics was determined by Kirby Bauer method. The inoculum concentrations were determined by Mc Farland method. BASE.128 and BASE as control (ALCHIMIA Srl) were inoculated with 10E5-10E6 cfu/ml of the selected microorganisms, each assessed in triplicate. All the strains were incubated at 37°C for 24 h, E. coli and E. faecalis were additionally incubated at +4°C for 48h and +22°C for 48h. Time kill of microorganisms by BASE.128 was determined at 0h, 3h, 6h, 16h, 24h (48h only for incubation at +4°C and 22°C).

Result: E. faecalis was resistant to cefuroxime, cetoconazol, clindamycin, quinupristin+dalfopristin, trimetoprim+sulfametoxazol. B. fragilis was penicillin resistant. A complete elimination of E. coli (6.56 log) was observed after 3h of incubation in BASE.128 at 37°C and 22°C and after 48h at 4°C. The initial inoculum of E. faecalis (5.7 log) was completely eliminated after 16h at 37°C, and 48h at 22°C. 5 log of F. magna and 6.6 log of P. melaninogenica were completely eliminated after 16h at 37°C. B. fragilis was reduced of 2.37 log after 16h at 37°C.

Conclusion: Decontamination with BASE.128 at 37°C for 16h allowed a complete elimination of at least 5 log of the most frequent umbilical cord contaminants (E. faecalis, E. coli, P. melaninogenica and F. magna).

Abstract: We investigated the presence of antibiotics in cryopreserved cardiovascular tissues and cryopreservation media, after tissue decontamination with antibiotic cocktails, and the impact of antibiotic residues on standard tissue bank microbiological analyses. Sixteen cardiovascular tissues were decontaminated with bank-prepared cocktails and cryopreserved by two different tissue banks according to their standard operating procedures. Before and after decontamination, samples underwent microbiological analysis by standard tissue bank methods. Cryopreserved samples were tested again with and without the removal of antibiotic residues using a RESEP tube, after thawing. Presence of antibiotics in tissue homogenates and processing liquids was determined by a modified agar diffusion test. All cryopreserved tissue homogenates and cryopreservation media induced important inhibition zones on both Staphylococcus aureus- and Pseudomonas aeruginosa-seeded plates, immediately after thawing and at the end of the sterility test. The RESEP tube treatment markedly reduced or totally eliminated the antimicrobial activity of tested tissues and media. Based on standard tissue bank analysis, 50% of tissues were found positive for bacteria and/or fungi, before decontamination and 2 out of 16 tested samples (13%) still contained microorganisms after decontamination. After thawing, none of the 16 cryopreserved samples resulted positive with direct inoculum method. When the same samples were tested after removal of antibiotic residues, 8 out of 16 (50%) were contaminated. Antibiotic residues present in tissue allografts and processing liquids after decontamination may mask microbial contamination during microbiological analysis performed with standard tissue bank methods, thus resulting in false negatives.

Abstract: Residual antibiotic concentrations may induce bacteriostasis of microorganisms eventually present on tissue samples and lead to false negative results during microbiological analysis of the sample.
The aim of the study was to validate RESEP for removal of antibiotic residues from solid and liquid samples before microbiological analysis.
The amount of antibiotics in 3-5 ml samples of four bank-prepared antibiotic cocktails, BASE.128 (AL.CHI.MI.A. Srl), cardiovascular, skin, amniotic membrane and corneal tissue samples was determined by HPLC and agar diffusion analyses, before and after treatment with RESEP. Interference of the device with bacterial growth was evaluated performing the microbiological recovery test with inoculants containing 1-10, 10-100 and 100-1000 CFU of European Pharmacopoeia (EP) reference strains (S. Aureus, P. Aeruginosa, C. Albicans, B. Subtilis, A. Niger and C. Sporogenes). Additional tests of the device were performed using different fluids and tissue samples provided by five different tissue banks.
Agar diffusion test and HPLC showed important antibiotic residues in liquid and tissue samples not treated with the RESEP; the amount of antibiotic residues varied depending on the antibiotic cocktail. Complete antibiotic removal from liquid and tissue homogenates decontaminated with BASE.128 was observed after RESEP treatment. Removal of antibiotics from bank-prepared antibiotic cocktails varied from 94-99%.
For EP reference strains, growth was observed for all the tested inoculants.
RESEP treated samples showed significantly higher bacterial recovery as compared to tissues analysed with traditional microbiological methods.
In conclusion, RESEP was validated for the removal of antibiotic residues from both liquid and tissue samples, thus allowing  detection of microbial growth.
The device resulted to be harmless for EP reference strains and showed higher sensibility as compared to currently used microbiological methods in tissue banks.

Abstract: Our previous studies indicated that the use of antibiotic cocktails during tissue processing can lead to an antibiotic carry-over effect, which in turn can generate false negative results in microbiological analysis.

Purpose: To validate a single-use device for elimination of antibiotics from liquid samples undergoing microbiological analysis to prevent interference with microbial growth.

Methods: The efficacy of the ResEP device was tested on 5 ml of BASE.128 (AL.CHI.MI.A.S.r.l.) and determined by HPLC after 5, 10, 20, 30 min. The recovery of S. Aureus, P. Aeruginosa, C. Albicans, B. Subtilis, A. Niger and C. Sporogenes was determined by dilution plating of inoculants containing 1-10, 10-100 and 100-1000 CFU, 20 min after treatment.

The device was further tested by three cardiovascular and skin Italian banks. Standard bacteriological tests were performed on transport, rinsing and cryopreservation liquids (BACT-ALERT Biomerieux or thioglycollate/TSB media) of twenty tissue samples decontaminated with BASE.128; additional bacteriological tests were performed on liquid samples using ResEP device, after antibiotic removal. The presence of antibiotic residues in all samples undergoing the microbiological analyses was evaluated by HPLC.

Results: The HPLC analysis of the samples treated with ResEP device showed complete antibiotic removal from the BASE.128 within 20 min of treatment. A total bacterial recovery was obtained for all investigated inoculants.
HPLC analysis on processing liquids showed that important antibiotic residues were present in rinsing and cryopreservation liquids, after decontamination of skin and cardiovascular tissues. The ResEP device removed efficiently antibiotic residues from all liquid samples prior to microbiological analysis allowing the detection of 10 % of false negative results.

Conclusion: The ResEP device was validated for easy and quick removal of antibiotic residues from liquid samples undergoing microbiological analysis, showing complete microbial recovery and high specificity for BASE.128 medium and allowing the detection of false negative results in microbiological analyses.

Abstract: Each tissue bank validates its own method for microbiological analysis of tissues, which is essential to determine whether they can be released for transplantation.
The aim of the study was to compare the results of microbiological analyses of tissues intended for transplantation obtained by using different microbiological testing methods.
Human cardiovascular tissues, skin and corneas were retrieved and processed by five different tissue banks. Tissues were decontaminated at 4°C for 24h/72h (cardiovascular) or at 22°C for 90 min. (skin) either with bank prepared solutions or BASE.128 (AL.CHI.MI.A. Srl., Italy) and then cryopreserved in RPMI 1640 with the addition of 10% DMSO. Corneas were processed and stored under organ culture conditions. Microbiological analysis of tissues and processing liquids were performed by tissue banks according to their standard procedures, using BacT/ALERT, BACTEC and direct inoculation of culture media (Thioglycollate/TSB); samples were tested in parallel at AL.CHI.MI.A. with the sterility test according to European Pharmacopoeia (EP), with the removal of antibiotic residues with RESEP.
All bacteriological analyses of decontaminated cardiovascular tissues performed with direct inoculum method resulted negative. 3% of liquid and tissue samples were found positive with the BACTEC method. The percentages of positive liquid and tissue samples with RESEP were 16% and 25%, respectively.
The bacteriological analysis of decontaminated skin samples were negative with both BACTEC and direct inoculum method. 33% of liquid samples and 50% of tissue samples were found positive using RESEP.
Corneas processing liquids resulted negative with BACTEC and direct inoculum method. Instead, 59% of the samples resulted positive with RESEP and sterility test according to EP.
Different results were obtained depending on the used method to perform microbiological analyses after tissue decontamination. A validation of the microbiological method, including accurate removal of possible antibiotic residues in decontaminated tissue and processing liquid is necessary to ensure the safety of tissue allografts.

Abstract: The presence of residual antibiotics in tissue allografts after decontamination with antibiotic cocktails may result in widely documented adverse effects in predisposed subjects.

Moreover, antibiotic residues may mask contaminating microorganisms, resulting in falsely negative sterility tests, with potential risk of post-surgical infections. The objective of the present study was to define a rinsing procedure capable of eliminating antibiotic residues from cardiovascular, bone and skin tissues after decontamination with BASE.128. Different washing patterns, employing BASE medium, were applied.

The presence of antibiotic residues in tissue homogenates was assessed by agar diffusion test at different stages of tissue processing. To test whether antibiotic residues can result in falsely negative microbiological analysis, we induced a superficial tissue contamination with known inoculum concentration. By employing four different porcine tissues, we here report direct evidence that the presence of even limited amounts of antibiotics in decontaminated tissues interferes with sterility testing. This has implications in terms of increased risk of infections in allograft recipients.

To minimize this risk, we developed a procedure for extensive removal of antibiotics from allografts, allowing for subsequent detection of microbial contaminations that may occur during transportation, storage or processing prior to allograft transplantation. Our study emphasizes the importance of validating all processes and analytical methods in tissue banking, in order to warrant tissue safety.

This will minimize the risks of post-surgical infections as well as antibiotic-induced anaphylaxis in predisposed patients.

Abstract: Decontamination of tissue allografts in antibiotic cocktails can lead to an antibiotic carry-over effect, which in turn can result in false negative in microbiological tests. Our investigation on the impact of antibiotic residues on microbiological analysis showed that a significant number of false negative results are found in tissues and liquid samples undergoing bacteriological analysis after decontamination. Our results suggest that both validation of the decontamination process and microbiological method is necessary to warrant  tissue safety.

Abstract: Skin decontamination and cryopreservation can result in the presence of antibiotic and glycerol residues, which shall be removed before transplantation  to ensure tissue safety. Our study showed that the elimination of antibiotic residues from cryopreserved skin depends on the type of antibiotic, thus suggesting that specific rinsing procedures shall be established and validated for each antibiotic solution used to decontaminate tissue.